PRODUCT MONOGRAPH

GEN-TAMOXIFEN

Tamoxifen Citrate Tablets

10 mg and 20 mg

Antineoplastic GENPHARM INC. Date of Preparation: 37 Advance Road May 8, 1990 Etobicoke, Ontario M8Z 2S6

CONTROL# 083764

Date of Revision: September 8, 2003

NAME OF DRUG

GEN-TAMOXIFEN

(Tamoxifen Citrate Tablets) 10 mg and 20 mg

ACTIONS AND CLINICAL PHARMACOLOGY

GEN-TAMOXIFEN (tamoxifen citrate) is a potent nonsteroidal derivative with antiestrogenic characteristics. It exerts its antiestrogenic effects by interfering with estrogen at its binding sites in such tissues as the uterus and the breast. Rat mammary carcinoma induction by dimethylbenzanthracene (DMBA) is inhibited by tamoxifen which is also effective against already established DMBA-induced tumours. This antitumour effect of tamoxifen appears to be mediated through estrogen receptor binding. Studies on human tissues revealed competitive binding between tamoxifen and estradiol at estrogen receptor proteins in breast and uterine adenocarcinomas and endometrium. Reports of advanced breast cancer trials conducted world-wide, however, indicate that, using established criteria, there is an objective response rate (complete and partial remission) to tamoxifen of approximately 10% in patients with estrogen receptor negative tumors which may indicate other mechanisms of action. An additional few patients experienced a stabilization of their disease. These results may be due to problems with the assay method, collection, transport or storage of tumour specimens affecting the estrogen receptor assay's validity. Also there can be great variability in amount of receptor protein (from 0 to 300 fmol/mg) in histologically comparable parts of one tumour (See ESTROGEN RECEPTOR ASSAY). Another explanation may be that tamoxifen is acting at sites apart from the estrogen receptors, as suggested by in vitro study results. Tamoxifen citrate has been found to have a low affinity for androgen receptors and a binding site separate from the estrogen receptor. Tamoxifen may also have an effect on cell membranes thereby altering the action of peptide hormones at their receptors. It may also affect cell growth by interfering with the action of steroid hormones or possibly limit tumour growth by inhibiting prostaglandin synthetase activity. It is recognized that tamoxifen also displays estrogenic-like effects on several body systems including the endometrium, bone and blood lipids.

CONTRAINDICATIONS

GEN-TAMOXIFEN (tamoxifen citrate) is contraindicated during pregnancy and in patients who have shown a hypersensitivity to the compound. Pre-menopausal patients must be examined to rule out the possibility of pregnancy prior to treatment. Tamoxifen must not be given during pregnancy. There have been a small number of reports of spontaneous abortions, birth defects and fetal deaths after women have taken tamoxifen, although no causal relationship has been established. Reproductive toxicology studies in rats, rabbits and monkeys have shown no teratogenic potential. In rodent models of fetal reproductive tract development, tamoxifen was associated with changes similar to those caused by estradiol, ethynylestradiol, clomiphene and diethylstilboestrol (DES). Although the clinical relevance of these changes is unknown, some of them, especially vaginal adenosis, are similar to those seen in young women who were exposed to DES in utero and who have a 1 in 1000 risk of developing clear cell carcinoma of the vagina or cervix. Only a small number of pregnant women have been exposed to tamoxifen. Such exposure has not been reported to cause subsequent vaginal adenosis or clear cell carcinoma of the vagina or cervix in young woman exposed in utero to tamoxifen. Women should be advised not to become pregnant while taking tamoxifen and should use barrier or other non-hormonal contraceptive methods if sexually active. Premenopausal patients must be carefully examined before treatment to exclude the possibility of pregnancy. Women should be appraised of the potential risks to the fetus, should they become pregnant while taking tamoxifen or within two months of cessation of therapy. When used in the prevention setting (an indication not approved in Canada) tamoxifen is contraindicated in patients with a history of stroke, deep vein thrombosis or pulmonary embolism, and in patients who are at an increased risk of developing endometrial cancer.

WARNINGS

GEN-TAMOXIFEN (tamoxifen citrate) should only be used for the conditions listed under the INDICATIONS section. Disturbances of menstrual cycle, including oligomenorrhea and amenorrhea, have been reported in some pre-menopausal women receiving tamoxifen citrate for the treatment of breast cancer. Although women receiving tamoxifen citrate for up to two years for the treatment of early breast cancer may develop disturbances of menstrual function on treatment, a proportion return to normal cyclical bleeding on termination of therapy. Hepatocellular carcinomas have been reported in a 2 year oncogenicity study in rats receiving tamoxifen (see TOXICOLOGY). In addition, gonadal tumors have been reported in mice receiving tamoxifen in long-term studies (see TOXICOLOGY). The clinical relevance of these cancer findings has not been established. A number of second primary tumors, occurring at sites other than the endometrium and the opposite breast, have been reported in clinical trials, following the treatment of breast cancer patients with tamoxifen. No causal link has been established and the clinical significance of these observations remains unclear. There is a possibility that tamoxifen citrate may cause tumours in animals and therefore the chance that this may also occur in humans must be noted. Cataracts were also reported in the 2 year oncogenicity study in rats, and since then it has been established that treatment with tamoxifen has been associated with an increased incidence of cataracts. An increased incidence of uterine malignancies has been reported in association with tamoxifen treatment. The underlying mechanism is unknown, but may be related to the estrogen-like effect of tamoxifen. Most uterine malignancies seen in association with tamoxifen are classified as adenocarcinoma of the endometrium. However, rare uterine sarcomas, including malignant mixed Mullerian tumors, have also been reported. Uterine sarcoma is generally associated with a higher FIGO stage (III/IV) at diagnosis, poorer prognosis, and shorter survival. Uterine sarcoma has been reported to occur more frequently among long-term users ($ 2 years) of tamoxifen than non-users. There is evidence of an increased incidence of thromboembolic events, including deep vein thrombosis and pulmonary embolism, during tamoxifen therapy. When tamoxifen is co-administered with chemotherapy, there may be a further increase in the incidence of thromboembolic effects. For treatment of breast cancer, the risks and benefits of tamoxifen should be carefully considered in women with a history of thromboembolic events. An increased risk of stroke has been found to be associated with tamoxifen therapy in high-risk patients being treated for the prevention of breast cancer. The use of tamoxifen for the prevention of breast cancer is not an approved indication in Canada. Incidence rates for the above events were estimated from a long-term clinical study called the National Surgical Adjuvant Breast and Bowel Project Breast Cancer Prevention (NSABP P-1) Trial. In this trial, high-risk patients were randomized to either tamoxifen therapy or placebo, for the prevention of breast cancer. Uterine malignancies were separated into cases of endometrial adenocarcinomas and uterine sarcomas. The relative risk of tamoxifen compared to placebo was 3.1 for endometrial cancer, 4.0 for uterine sarcomas, 1.6 for stroke, 3.0 for pulmonary embolism, and 1.6 for deep vein thrombosis.

PRECAUTIONS

In some instances, a temporary decrease in platelet and leukocyte counts have been measured during treatment. While these changes may or may not have been caused by tamoxifen and no hemorrhagic tendency has been reported, complete blood counts, including platelet counts, are recommended at regular intervals. Tamoxifen citrate should be used with care in patients with thrombocytopenia or leukopenia. During treatment with tamoxifen, platelet counts have decreased temporarily to values between 50,000 and 100,000/mm3. On rare occasions even lower counts have been observed without hemorrhagic tendencies. Treatment was not interrupted and platelet counts were reported to have returned to normal. The known principal pathway for tamoxifen metabolism in humans is demethylation catalysed by CYP3A4 enzymes. A pharmacokinetic interaction with the CYP3A4 inducing agent rifampicin, involving a reduction in tamoxifen plasma levels has been reported in the literature. The relevance of this to clinical practice is not known. Breast cancer patients with metastatic bone disease may develop hypercalcemia within a few weeks of treatment and therefore must be monitored regularly for serum calcium levels or other signs of hypercalcemia. In the presence of severe hypercalcemia, treatment should be discontinued, whereas in less severe cases appropriate measures should be taken. The first prognostic follow-up of the patient after beginning treatment with GEN-TAMOXIFEN (tamoxifen citrate) should be done within the first four weeks, with subsequent examinations done at four to eight week intervals. Adverse reactions like hot flashes, nausea, and vomiting can be controlled in some patients by dose reductions without reducing the effect on the disease. If patients experience bone pain, analgesics may be administered. An increased incidence of endometrial cancer and uterine sarcoma (mostly malignant mixed Mullerian tumors) has been reported in association with tamoxifen treatment. The incidence and pattern of this increase suggest that the underlying mechanism may be related to estrogenic properties of tamoxifen. Any patients receiving tamoxifen or having previously received tamoxifen who report abnormal gynaecological symptoms, especially vaginal bleeding, should be promptly investigated. Clinical studies have shown a median treatment duration of two months before an identifiable response was observed. It should also be noted that in 25% of the patients it required a minimum of four months before an objective response was observed. Treatment with tamoxifen is recommended as long as it is effective. Although the drug should be discontinued if there is obvious disease progression, it is suggested that initial treatment be continued for at least three or four weeks if possible, since occasional patients have local disease flares or increased bone pain early on in treatment. This can make it difficult to determine, at that stage, whether the disease will continue to progress, stabilize or respond to continued tamoxifen administration.

Drug Interactions:

When tamoxifen is used in combination with coumarin-type anticoagulants, a significant increase in anticoagulant effect may occur. Where such coadministration exists, careful monitoring of the patient's prothrombin time is recommended.

When tamoxifen is used in combination with cytotoxic agents, there is increased risk of thromboembolic events occurring.

Lactation:

It is not known if tamoxifen is excreted in human milk and, therefore, the drug is not recommended during lactation. The decision either to discontinue nursing or discontinue tamoxifen should take into account the importance of the drug to the mother.

PHARMACOLOGY

Pharmacokinetics and Metabolism

Maximum serum levels in women after a single oral 20 mg dose of tamoxifen were achieved in 3 to 7 hours, reaching peak levels of 0.06 to 0.14 :g/mI with only 20-30% of the drug present as tamoxifen. The distribution half-life was reported to be 7 to 14 hours with secondary peaks 4 or more days later. Using radiolabelled tamoxifen, most of the radioactivity was slowly excreted in the feces, with only small amounts present in the urine. Tamoxifen is excreted mainly as conjugated metabolites. The parent drug and hydroxylated metabolites account for 30% of the total. Tamoxifen undergoes enterohepatic recirculation, which may explain the prolonged blood levels and relatively high fecal excretion.

Antiestrogenic Effect

The antiestrogenic effects of tamoxifen are exerted in spayed and immature rats by inhibiting estrogen induced vaginal cornification and uterine growth, respectively. Tamoxifen is also able to prevent the implantation of the blastocysts in rats, thereby terminating early pregnancy. It competes with the estrogen released by the ovaries on day 4 of pregnancy needed for implantation on day 5. Ovulation in female rats with regular estrous cycles can be delayed with a single dose of tamoxifen administered on or before the day of diestrous. In rats and other species that ovulate spontaneously, it is the action of estrogen on the hypothalamus and/or pituitary which then causes the pituitary to release the luteinizing hormone (LH). Although the secretion of estrogen from the ovaries reaches a peak before this LH discharge, the inhibitory effect of tamoxifen on ovulation is attributed to interference with "feedback" action of estrogen at the hypothalamic and/or pituitary level. Tamoxifen is also antiestrogenic in the pig-tailed monkey (M. nemestrina). In regularly menstruating mature females of this species (intervals of about 28 days) there is an edematous swelling of the perineal region during the follicular phase which goes away more quickly during the period of ovulation. This swelling is caused by estrogen and such an effect does not occur in ovariectomized monkeys unless they receive estrogen. Large daily doses of tamoxifen alone to ovariectomized female pig-tails caused no perineal region swelling. In such animals, small oral doses of tamoxifen was able to reduce the swelling caused by daily injections of estradiol almost to zero. While tamoxifen is able to inhibit the effects of estrogen in spayed rats and monkeys, it may also inhibit the endogenous estrogen production in intact animals. Tamoxifen, in very large doses, also has some estrogenic activity as seen by incomplete vaginal cornification and limited increased uterine weight in spayed rats. In mice, tamoxifen has estrogenic properties without demonstrable estrogen antagonistic activity at any dose.

Bioavailability:

The relative bioavailability of GEN-TAMOXIFEN (20 mg) tablets of Genpharm Inc. Canada and NolvadexR (20 mg) tablets of ICI Pharma Canada was compared. The study was a single dose (2 x 20 mg of each brand) administered to each of 16 fasted healthy volunteers in a balanced randomized crossover design with a 6 week washout period. Blood samples were collected up to 1008 hours. Both tamoxifen and N-desmethyltamoxifen were assayed in the plasma using a validated HPLC method. The results of this biostudy are summarized in the following table: Mean Pharmacokinetic Data (+- SD)

Complete details on the comparative bioavailability study are available from Genpharm Inc. Canada upon request.

TOXICOLOGY

Acute Toxicity Studies

In all species studied (mice, rats, rabbits and marmosets), tamoxifen citrate has shown a low acute toxicity with an oral LD50 greater than 1 g/kg. In one study, the oral LD50 in mice was 3100 mg/kg whereas the intraperitoneal LD50 was 270 mg/kg. In another study, the oral LD50s in mice and rats were found to be 3 and 2.5 g/kg, respectively, and the intravenous LD50 for both species was 62.5 mg/kg.

Long Term Toxicity Studies:

Rats, dogs and marmosets were used to conduct long term toxicity studies. In a three month rat study, tamoxifen was administered as a mixture made up of approximately 10% of the corresponding cis-isomer (an estrogen). Daily doses of 2, 20 and 100 mg/kg of the mixture resulted in a reduction (relative to body weight) in the weight of testes, seminal vesicles, ventral prostate and ovaries. There was also a reduction in the number of corpora lutea and follicular cysts, and in uterine size. In all treated rats, there was a complete absence of glands in the endometrium with the epithelium consisting of a single layer of columnar cells with occasional squamous metaplasia and small areas of flattening. The endometrial stroma was fibrous in appearance due to it being somewhat condensed. Male rats which received low doses had decreased numbers of spermatocytes and occasional atrophic tubules. At the intermediate dosage, changes were similar but not as severe. In male rats receiving high doses, there were scattered necrotic cells in the seminiferous epithelium and all maturation of spermatozoa stopped. The height of the thyroid epithelium was increased slightly in a few treated rats whereas a thin zone of adrenal cortical congestion and edema was noted in all of the treated rats. Using the same cis-trans mixture as was used in the three month rat study, oral doses of 1, 10 and 50 mg/kg tamoxifen were studied in dogs for three months. A decrease in the weight of the pituitary and testes was observed in the treated males in all groups, while females showed an increase in uterine weight. Cessation of ovulation, decreased numbers of follicles and hyperplasia of the germinal epithelium were observed in the ovaries of the treated females. Hyperplasia of the germinal epithelium is an exaggeration of the physiological changes seen in metestrus. In dogs receiving the lower doses these changes were not as extensive. Squamous metaplasia of the endometrium and severe endometritis were observed in the uterus of all treated females. Although changes in the total bulk of uterine muscle seemed unlikely, the myometrium showed separation of the muscle bundle by an obvious edematous connective tissue causing the muscle to have an "attenuated" appearance. In most tubules, the seminiferous epithelium consisted of only one layer of spermatogonia and Sertoli cells. Due to atrophy caused by the estrogenic effect of the cis-trans mixture, there was a remarkable increase in the fibrous stroma around the tubules resulting from the condensation of the normal interstitial tissue. All treated dogs had atrophic testes. In the highest dosage group, the livers of three males and one female had pigment in the Kupffer cells and bile plugs in the bile canaliculi. Except for some slight thinning of the cell cords, the liver was normal which is consistent with the biochemical effect of raised serum alkaline phosphatase. It should be noted, that the dose in this case is 500 times that needed to prevent implantation in the dog. All other organs showed changes which were within normal limits. Orally administered tamoxifen in doses of 0.05, 0.8, 2.4, 4.8 and 9.6 mg/kg to rats for a period of 6 months showed that only the three highest doses resulted in some changes. These changes included severe atrophic changes in the reproductive organs which increased with doses from 2.4 to 9.6 mg/kg, increased levels of serum alkaline phosphatase and sodium as well as decreased alanine amino transferase, aspartate aminotransferase and albumin levels. There were no significant histological findings observed in the liver. In an on-going two year study, rats were administered 5, 20 and 35 mg/kg tamoxifen by gavage (all of which represent significant multiples of the recommended human dose of 20-40 mg/day). The changes noticed by the 15th month, included atrophy of the reproductive organs along with hepatocellular carcinomata in the 20 and 35 mg/kg dose groups. Also a dose-related increase in cataracts was noted. In another study, lasting 29 days, 12 rats/sex/dose were administered 0 and 100 mg/kg tamoxifen. All females and 3 male rats died within the first 2 weeks. Toxic symptoms included bleeding from nose, piloerection and weakness. In male rats, slight leukopenia, thrombocytopenia and anemia were noted. Enlarged adrenals and smaller thymus and seminal vesicles were observed at autopsy. In another study, lasting 3 months, rats were given 0.35 and 70 mg/kg. At both dose levels, 50% of the females and 17% of the males died. An increase in the food intake, nose bleeding and decreased locomotor activity were noted. An increase in adrenal size and a decrease in genital size were observed at autopsy. Histopathology showed atrophy of reproductive system and hyperplasia of adrenal cortex. Slight anemia and thrombocytopenia were also noted. In a 3 month study, dogs receiving oral doses of up to 75 mg/kg tamoxifen, were found to have effects primarily on the reproductive system. At the highest dose, biliary stasis was also seen. In a 6 month study involving marmosets, tamoxifen was orally administered in doses of 0.8, 4.0 and 8.0 mg/kg. The formation of cystically enlarged follicles in the ovaries of the females treated with the 8.0 mg/kg dose was the only treatment related, pathologically significant effect noted. The pure cis and trans-isomers at an oral dose of 20 mg/kg were compared in an additional two month study in rats. The activity of tamoxifen was similar with respect to the aforementioned reproductive tissue changes for all treatment groups. However, the adrenal and thyroid lesions were seen only in those rats receiving the cis-isomer. In female rats, a reversibility test was conducted using tamoxifen citrate administered orally at doses of 0.5 and 2.0 mg/kg for three months where one third of the animals were not given tamoxifen for an additional three months. After three months treatment, the ovaries and uteri showed changes similar to those described above but were reversible in the rats kept without dosing for an additional three months. Tamoxifen citrate was compared with stilboestrol and clomiphene in a reversibility study using female dogs. When testing for reversibility, the 0.1 mg/kg dose of tamoxifen citrate was administered for three months with one animal out of four remaining untreated for another month. The myometrium showed a decrease of collagen with fragmentation of the bundles. Edema was present and caused separation of the muscle bundles. Squamous metaplasia did not develop in the uterus of dogs given tamoxifen citrate. Upon withdrawal of tamoxifen citrate, there was increased collagen produced in thick bundles, an effect similar to a slight estrogenic change. Hyperplasia of the germinal epithelium occurred in the ovaries along with cessation of ovulation. In studies comparing tamoxifen with conventional estrogen, gonadal tumours were present in mice, an effect which could be attributed to the estrogenic activity of tamoxifen. The cis-trans mixture described above was administered orally in mice at doses of 5 and 50 mg/kg for fifteen months. Following this, the pure cis and trans forms were compared with the cis-trans mixture at a dose of 20 mg/kg and with

stilboestrol and ethinyl estradiol for a period of thirteen months. To investigate the effects of lower doses of the cis, trans and cis-trans mixture of tamoxifen with stilboestrol and ethinyl estradiol, an additional study of fourteen months duration was conducted. The dosage used was 0.1 mg/kg. The results showed that the compound produced interstitial cell tumours of the testes and granulosa cell tumours of the ovary. Spinal deformity with kyphosis, characterized as elongation of vertebral bodies, developed in mice after six months of treatment. Due to ossification of the medullary cavity, there was increased opacity of long bone. Estrogenic activity of tamoxifen is the cause of some of these changes; the etiology of others remained unknown and were not seen at lower doses. To assess the ocular toxicity of tamoxifen citrate, three tests were performed in which compounds known to cause ocular lesions and having chemical structures similar to tamoxifen such as clomiphene and triparanol were compared. In the first two tests, mated female rats were dosed on day 11 of pregnancy, with tamoxifen citrate, clomiphene B or clomiphene. The animals were sacrificed on day 19 or 20. In addition to observations on the uterine and fetal changes, the eyes of the fetuses were then examined histologically. In the third experiment, pregnant females were given clomiphene on day 11 of pregnancy and the fetuses were removed by cesarean section on day 22. After immediate fostering to control animals, to allow development to weaning, they were sacrificed and examined for cataracts. No significant increases in embryonic or fetal deaths were noted in the first two studies in any of the treatment groups. The clomiphene and clomiphene B groups developed fetal cataracts and the tamoxifen citrate group did not. There was a 9.5% incidence of cataracts induced by clomiphene in fostered neonates in the third test. Treated rats developed an increase in placental weight and a decrease in uterine weight; hydramnios was also present.

Reproductive Studies:

In rats and rabbits, teratogenic studies were conducted with great difficulty since tamoxifen inhibits implantations. Doses used in rats ranged from 0.02 to 4.0 mg/kg orally and from 0.01 to 2.0 mg/kg in rabbits (administered in the feed). A reversible rib deformity which under certain conditions approached a 50% incidence was the only drug-induced abnormality detected and occurred only in rats. The cause of the deformity appears to be a mechanical one due to the failure of uterine growth, an antiestrogenic property of the compound. Tamoxifen is not mutagenic in a range of in vitro and in vivo mutagenicity studies. Tamoxifen was genotoxic in some in vitro tests and in vivo genotixicity tests in rodents.

ESTROGEN-RECEPTOR ASSAY

Introduction:

Recent studies in estrogen dependent tissues have shown that there is a cytoplasmic protein which binds estrogen with high affinity and specificity. Although estrogen enters the cytoplasm of all cells, an estrogen-receptor complex will only be formed in the cytoplasm of estrogen dependent cells. These cells contain specific protein molecules (receptors) in the cytoplasm which allow estrogen to exert its specific biological effect. Once estrogen is bound to the receptor, the estrogen-receptor complex undergoes an activation step allowing it to enter the nucleus of the cell and bind to chromatin, the genetic information of the cell. This in turn leads to messenger RNA production. The messenger RNA molecules are then released into the cytoplasm where they can be translated on polysomes into new protein. Since anti-estrogens can also enter the cell cytoplasm of the estrogen dependent cell, they can bind to the protein receptor with affinity and specificity, thereby activating the complex to translocate to the nucleus. However, the normal estrogen transcriptional processes are altered by the anti-estrogens. By competing with estrogens for the receptor site, the anti-estrogens interfere with estrogen-dependent tumour growth by shutting down the normal processes of the genetic information within the nucleus. The results of reports concerning the relationship between clinical responses of patients with breast cancer receiving endocrine therapy and the presence or absence of estrogen receptors have shown that approximately 56% of patients with estrogen-receptor positive tumours will respond to endocrine therapy. In contrast, only about 10% of patients respond to therapy if they have tumours which are negative for estrogen receptors. Thus in patients with breast cancer, estrogen receptor assays are useful in predicting a response to endocrine therapy.

Methods:

1. Dextran-Coated Charcoal Assay (DCC) In this assay, the highly labile estradiol receptor is extracted from a cytosol prepared from the tumour tissue. After incubating with tritiated estradiol, which interacts with receptor binding sites, the excess estradiol is separated from the incubate with dextran coated charcoal. The amount of nonspecific binding (such as albumin) is then assessed and the number of estradiol receptors in the tissue can be estimated by subtracting the nonspecific binding from the total binding per milligram of protein. Estrogen-receptor negative tumours show binding capacity similar to benign tumours. Estrogen-receptor positive tumours show higher binding capacity.

2. Sucrose Gradient Method (SG) A weighed tumour specimen is immersed in liquid nitrogen and shattered. The residual tissue powder is homogenized with efficient cooling in four volumes of buffer using a tissue disintegrator with two or three homogenization periods, each followed by a cooling period. To precipitate the particulate matter, the homogenate is centrifuged. Two portions of the cytosol fraction are removed and treated with either buffer alone or buffer containing an agonist. Tritiated estradiol is added to each mixture when equilibrium is reached. After mixing and standing in the cold, a portion of each mixture is layered on a 10 to 30% sucrose gradient containing buffer and centrifuged. Successive fractions are then collected, and the radio activity counted. An 8 S complex occurs with receptor-positive tumour specimens whereas different amounts of specific binding in the 4 S region occur with other specimens. From the difference in sedimentation curves, with and without inhibitor, from fraction 1 to the minimum observed (around fraction 18 to 22 depending on the ultracentrifugation), the radioactivity associated with the 8 S form of estrophilin is estimated. The 4 S radioactivity is similarly calculated by difference of the curves between the minimum and the point where the curve with inhibitor crosses the curve without inhibitor. Interpretation of Results Since laboratory results of the estrogen-receptor assay vary according to technique, handling and storage of specimen, and the menopausal status of the patient or recent drug therapy, only qualified experts should interpret results. Quantitative results vary between laboratories and methods. Based on patient response to hormonal manipulation, investigators have done retrospective correlations which show that a result of less than 3 fmoles/mg of cytosol protein is estrogen receptor-negative, 3 to 10 fmol/mg is equivocal, and greater than 10 fmoles/mg is considered estrogen-receptor positive. ESTROGEN RECEPTOR MONOCLONAL ANTIBODIES The quantitative determination of estrogen and progesterone receptors in human breast cancers has served as a guide to therapeutic invention as well as prognosis. At the time of mastectomy, the response to endocrine therapy, should the tumour recur, as well as the probability and rapidity of recurrence may be estimated by analyzing the receptor content of the primary tumours. Current methods for determinations of estrogen and progesterone receptors suffer from several deficiencies which include being expensive in terms of laboratory time, requiring a large sampling of tumour tissue, erroneous results from rapid receptor deterioration during specimen processing or storage, and failure of ligand-binding assays to detect receptors that are already complexed with non-radioactive hormone of endogeneous or therapeutic origin. These deficiencies have lead to investigations of improved techniques for a simple, accurate, and inexpensive assay which will recognize the receptor whether or not it retains its ability to bind hormones. A number of monoclonal antibodies specific for antigenic determinants on or near the estrogen receptor site has been generated by Greene and Jensen of the University of Chicago, Chicago, Illinois. The extranuclear estrogen receptor of the MCF-7 human breast cancer cell line are recognized by these specific monoclonal antibodies. These antibodies bind to nuclear and cytosolic estrogen receptors from a variety of tissues and are therefore unique and specific probes for examining the structure and function of the estrogen receptor. Different antigenic determinants on the receptor molecule are recognized by three antibodies, D58, D75 and D547. Two such antibodies can be used in a sandwich technique for the immunoradiometric (IRMA) or enzyme linked immunosorbent (ELISA) determination of estrogen receptor. These three antibodies recognized estrogen receptors in human breast cancer specimen as well as estrogen receptors in uterine tissue from other species. The D547 and D58 monoclonal antibodies have been noted to distinguish among various forms of the estradiol-estrogen receptor complex. The antigenic determinants recognized by these particular antibodies on breast tumour cytosolic receptors are not significantly altered by the binding of either estrogen or antiestrogen to the receptor. Studies such as this are able to demonstrate fundamental differences in the subcellular fate of the estrogen or antiestrogen-receptor complexes, and provide clues to the mechanism of action of estrogens and antiestrogens. An immunoperoxidase technique was used by Poulsen with two monoclonal antibodies specific for MCF- 7 estrogen receptor to stain human breast cancer tissue sections. The immunoperoxidase staining was predominantly located in the nucleus of the malignant epithelial cells. Although no relationship between tumour type or degree of differentiation of invasive ductal carcinomas and staining features was noted, Poulsen found a significant positive correlation between the number of positively stained cells and cytosol receptor content. Similarly, King has developed monoclonal antibody D-5, an lgG1 which binds specifically to human soluble estrogen receptor in a dose-dependent manner, and will not react with other steroid binding proteins or nuclear estrogen receptor. A highly significant correlation was noted by King between estrogen receptor content and D-5 reactivity in human breast cancer sections. Kodama, when using similar techniques to study the expression of estrogen receptors of human breast cancer clonal growth using the soft-agar cloning assay, found that estrogen receptor expression increased with clonal growth of tumour cells to colonies and that estrogen receptor appeared to be expressed in the differentiation process. Also Dr. Edwards has developed a monoclonal antibody to the chicken oviduct progesterone receptor which also recognizes denatured human progesterone receptor as its antigen. Further applications of this monoclonal antibody are presently being investigated. The development of specific monoclonal antibodies directed at antigenic determinants of the estrogen or progesterone receptor will make it possible to more accurately and precisely define levels of estrogen or progesterone receptors in human tumour tissue. 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Biol Reprod 1979; 21:1087-1090.Purr, B.J., et al. Tamoxifen. In: Goldberg, M.E. ed. Pharmacological and biochemical properties of drug substances. Vol 2 American Pharmacological Association, 1979; 355-399.Adam, H.K., J.S. Patterson and J.V. Kemp. Studies on the metabolism and pharmacokinetics of tamoxifen in normal volunteers. Cancer Treat Rep 1980; 64:761-764.Bloomer, W.D. et al. Iodine-125-labelled tamoxifen is differentially cytoxic to cells containing estrogen receptors. Int J Radiat Biol 1980; 38(2): 197-202.De Quijada, M. et al. Tamoxifen enhances the sensitivity of dispersed prolactin secreting pituitary tumour cells to dopamine and bromocriptine. Endocrinology 1980; 106(3):702-706.Greene, G.L., F.W. Fitch and E.V. Jensen. Monoclonal antibodies to estrophilin probes for the study of estrogen receptors. Proc NatI Acad Sci USA 1980; 77(1):157-161.Wilkinson, P. et al. Clinical pharmacology of tamoxifen and N-desmethyltamoxifen in patients with advanced breast cancer. Cancer Chemother Pharmacol 1980; 5:109-111.Campbell, F. C. et al. Quantitative oestradiol receptor values in primary breast cancer and response of metastases to endocrine therapy Lancet 1981; 1317-1319.Clark, J.H., S.C. Guthrie and S.A. McCormack. Neonatal stimulation of the uterus by clomiphene, tamoxifen and nefoxidine: relationship to the development of reproductive tract abnormalities. Adv Exp Med Biol 1981; 138:87-98.Fabian, C. et al. Clinical pharmacology of tamoxifen in patients with breast cancer: correlation with clinical data. Cancer 1981; 48:876-882.Legha, S.S. et. al. Tamoxifen-induced hypercalcemia in breast cancer. Cancer 1981; 47: 2803- 2806.Lullmann, H. and R. Lullmann-Rauch. Tamoxifen-induced generalized lipidosis in rats subchronically treated with high doses. Toxicol Appl Pharmacol 1981; 61:138-146.McKeown, C.A. et al. Tamoxifen retinopathy. Br J Ophthalmol 1981; 65:177-179.Antineoplastic agents and immunosuppressants. In: Reynolds, J.E.F. ed. Martindale, The Extra Pharmacopoeia, 28th Edition. London, The Pharmaceutical Press, 1982; 226-227.Bezwoda, W.R. et al. Treatment of metastatic breast cancer in estrogen receptor positive patients: a randomized trial comparing tamoxifen alone versus tamoxifen plus CMF . Cancer 1982; 50:2747-2750.Greene, G.L. and E.V. Jensen. Monoclonal antibodies as probes for estrogen receptor detection and characterization. J Steroid Biochem 1982; 16:353-359.Lipton,A. et al. Randomized trial of aminoglutethimide versus tamoxifen in metastatic breast cancer. Cancer Res 1982; 42(suppl):3434s-3436s.Pearson, O.H., A. Manni and B.M. Arafah. Antiestrogen treatment of breast cancer: an overview.Cancer Res 1982; 42(suppl): 3424s-3429s.Pugh,D.M. and H.S. Sumano. The anti-implantation action of tamoxifen in mice. Arch Toxicol 1982; Suppl 5:209-213.Bradbeer, J.W. and J. Kyngdon. Primary treatment of breast cancer in elderly women with tamoxifen. Clin Oncol 1983; 9:31-34.Ribeiro,G. and M.K. Palmer. Adjuvant tamoxifen for operable carcinoma of the breast: report of clinical trial by the Christie Hospital and Holt Radium Institute. Br Med J 1983; 286:827-830.Sutherland, R.L., R.E. Hall and l.W. Taylor. Cell proliferation kinetics of MCF-7 human mammary carcinoma cells in culture and effects of tamoxifen on exponentially growing and plateau-phase cells. Cancer Res 1983; 43:3998-4006.Vinding, T. and N.V. Nielsen. Retinopathy caused by treatment with tamoxifen in low dosage. Acta Opthalmol 1983; 61:45-50.Bratherton, D.G. et al. A comparison of two doses of tamoxifen in postmenopausal women with advanced breast cancer: 10 mg bd versus 20 mg bd. Br J Cancer 1984; 50:199-205.Ingle, J.N. Additive hormonal therapy in women with advanced breast cancer. Cancer 1984; 53(3):766-777Kodama, F. et al. Expression of estrogen receptor as a clonal marker of differentiation in MCF-7 cells. Am Assoc Cancer Res 1984; 25:218.Nemoto, T. et al. Tamoxifen (Nolvadex) versus adrenalectomy in metastatic breast cancer.Cancer 1984; 53:1333-1335.Reddel, R.R., L.C. Murphy and R.L. Sutherland. Factors affecting the sensitivity of T-47D human breast cancer cells to tamoxifen. Cancer Res 1984; 44:2398-2405.Taylor, C.M., B. Blanchard and D.T. Zava. Estrogen receptor-mediated and cytotoxic effects of the anti- estrogens tamoxifen and 4-hydroxytamoxifen. Cancer Res 1984; 44:1409-1414.Baum, M. et al. Controlled trial of tamoxifen as single adjuvant agent in the management of early breast cancer: analysis at six years by Nolvadex adjuvant trial organisation. Lancet 1985; 1:836- 840Ribeiro, G. and R. Swindell. The Christie Hospital tamoxifen (Nolvadex) adjuvant trial for operable breast carcinoma - 7 year results. Eur J Cancer Clin Oncol 1985; 21(8):897-900.Taguchi, O. and Y. Nishizuka. Reproductive tract abnormalities in female mice treated neonatally with tamoxifen. Am J Obstet Gynecol 1985; 151:675-678.Taylor, S.G. et al. Adjuvant CMFP versus CMFP plus tamoxifen versus observation alone in postmenopausal, node-positive breast cancer patients: three-year results of an eastern co- operative oncology group study. J Clin Oncol 1985; 3(2):144-154.Product Monograph. TamofenR (tamoxifen citrate) tablets of 10 and 20 mg. Rhone-Poulenc Pharma Inc. Montreal, Quebec, January 13.1988.Product Monograph. Nolvadex (tamoxifen citrate) Tablets 10 mg. Nolvadex-D (tamoxifen citrate) Tablets 20 mg. ICI Pharma, Mississauga, Ontario, June 14, 1988.United States Pharmacopeia. Drug information for the health care provider. Eighth Edition 1988; IB:2005-2006.Zelissen, P., M. A. Blankenstein and J. J. Tukker. Comparative bioavailability of tamoxifen citrate. Comparison of tamoxifen citrate GenericsR with Nolvadex-DR in 16 male volunteers. Data on file, Genpharm Inc., 1988.

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11. Jordan, V.C. Effect of tamoxifen (ICI 46,474) on initiation and growth of DMBA-induced rat mammary carcinomata. Eur J Cancer 1976; 12:419-424.

12. Kiang, D.J. and B.J. Kennedy. Tamoxifen (antiestrogen) therapy in advanced breast cancer. Ann Intern Med 1977; 87(6):687-690.

13. Heel, R.C. et al. Tamoxifen: a review of its pharmacological properties and therapeutic use in the treatment of breast cancer. Drugs 1978; 16:1-24.

14. Kaiser-Kupfer, M.I. and M.E. Lippman. Tamoxifen retinopathy. Cancer Treat Rep1978; 62(3):31 5-320.

15. Jordan, V.C. and K.E. Naylor. The antitumour activity of tamoxifen and monohydroxytamoxifen: a comparative study in the rat. Br J Pharmacol 1 978; 63:376P-377P.

16. Patterson, J.S. and M. Baum. Safety of tamoxifen. Lancet 1978; 1(8055): 105.

17. Ward, H.W.C. et al. Anti-oestrogen therapy for breast cancer - a report on 300 patients treated with tamoxifen. Clin Oncol 1978; 4:11-17.

18. Chamness, G.C. et al. Abnormal reproductive development in rats after neonatally administered antiestrogen (tamoxifen). Biol Reprod 1979; 21:1087-1090.

19. Purr, B.J., et al. Tamoxifen. In: Goldberg, M.E. ed. Pharmacological and biochemical properties of drug substances. Vol 2 American Pharmacological Association, 1979; 355-399.

20. Adam, H.K., J.S. Patterson and J.V. Kemp. Studies on the metabolism and pharmacokinetics of tamoxifen in normal volunteers. Cancer Treat Rep 1980; 64:761-764.

21. Bloomer, W.D. et al. Iodine-125-labelled tamoxifen is differentially cytoxic to cells containing estrogen receptors. Int J Radiat Biol 1980; 38(2): 197-202.

22. De Quijada, M. et al. Tamoxifen enhances the sensitivity of dispersed prolactin secreting pituitary tumour cells to dopamine and bromocriptine. Endocrinology 1980; 106(3):702-706.

23. Greene, G.L., F.W. Fitch and E.V. Jensen. Monoclonal antibodies to estrophilin probes for the study of estrogen receptors. Proc NatI Acad Sci USA 1980; 77(1):157-161.

24. Wilkinson, P. et al. Clinical pharmacology of tamoxifen and N-desmethyltamoxifen in patients with advanced breast cancer. Cancer Chemother Pharmacol 1980; 5:109-111.

25. Campbell, F. C. et al. Quantitative oestradiol receptor values in primary breast cancer and response of metastases to endocrine therapy Lancet 1981; 1317-1319.

26. Clark, J.H., S.C. Guthrie and S.A. McCormack. Neonatal stimulation of the uterus by clomiphene, tamoxifen and nefoxidine: relationship to the development of reproductive tract abnormalities. Adv Exp Med Biol 1981; 138:87-98.

27. Fabian, C. et al. Clinical pharmacology of tamoxifen in patients with breast cancer: correlation with clinical data. Cancer 1981; 48:876-882.

28. Legha, S.S. et. al. Tamoxifen-induced hypercalcemia in breast cancer. Cancer 1981; 47: 2803- 2806.

29. Lullmann, H. and R. Lullmann-Rauch. Tamoxifen-induced generalized lipidosis in rats subchronically treated with high doses. Toxicol Appl Pharmacol 1981; 61:138-146.

30. McKeown, C.A. et al. Tamoxifen retinopathy. Br J Ophthalmol 1981; 65:177-179.

31. Antineoplastic agents and immunosuppressants. In: Reynolds, J.E.F. ed. Martindale, The Extra Pharmacopoeia, 28th Edition. London, The Pharmaceutical Press, 1982; 226-227.

32. Bezwoda, W.R. et al. Treatment of metastatic breast cancer in estrogen receptor positive patients: a randomized trial comparing tamoxifen alone versus tamoxifen plus CMF . Cancer 1982; 50:2747-2750.

33. Greene, G.L. and E.V. Jensen. Monoclonal antibodies as probes for estrogen receptor detection and characterization. J Steroid Biochem 1982; 16:353-359.

34. Lipton,A. et al. Randomized trial of aminoglutethimide versus tamoxifen in metastatic breast cancer. Cancer Res 1982; 42(suppl):3434s-3436s.

35. Pearson, O.H., A. Manni and B.M. Arafah. Antiestrogen treatment of breast cancer: an overview. Cancer Res 1982; 42(suppl): 3424s-3429s.

36. Pugh,D.M. and H.S. Sumano. The anti-implantation action of tamoxifen in mice. Arch Toxicol 1982; Suppl 5:209-213.

37. Bradbeer, J.W. and J. Kyngdon. Primary treatment of breast cancer in elderly women with tamoxifen. Clin Oncol 1983; 9:31-34.

38. Ribeiro,G. and M.K. Palmer. Adjuvant tamoxifen for operable carcinoma of the breast: report of clinical trial by the Christie Hospital and Holt Radium Institute. Br Med J 1983; 286:827-830.

39. Sutherland, R.L., R.E. Hall and l.W. Taylor. Cell proliferation kinetics of MCF-7 human mammary carcinoma cells in culture and effects of tamoxifen on exponentially growing and plateau-phase cells. Cancer Res 1983; 43:3998-4006.

40. Vinding, T. and N.V. Nielsen. Retinopathy caused by treatment with tamoxifen in low dosage. Acta Opthalmol 1983; 61:45-50.

41. Bratherton, D.G. et al. A comparison of two doses of tamoxifen in postmenopausal women with advanced breast cancer: 10 mg bd versus 20 mg bd. Br J Cancer 1984; 50:199-205.

42. Ingle, J.N. Additive hormonal therapy in women with advanced breast cancer. Cancer 1984; 53(3):766-777

43. Kodama, F. et al. Expression of estrogen receptor as a clonal marker of differentiation in MCF-7 cells. Am Assoc Cancer Res 1984; 25:218.

44. Nemoto, T. et al. Tamoxifen (Nolvadex) versus adrenalectomy in metastatic breast cancer. Cancer 1984; 53:1333-1335.

45. Reddel, R.R., L.C. Murphy and R.L. Sutherland. Factors affecting the sensitivity of T-47D human breast cancer cells to tamoxifen. Cancer Res 1984; 44:2398-2405.

46. Taylor, C.M., B. Blanchard and D.T. Zava. Estrogen receptor-mediated and cytotoxic effects of the anti- estrogens tamoxifen and 4-hydroxytamoxifen. Cancer Res 1984; 44:1409-1414.

47. Baum, M. et al. Controlled trial of tamoxifen as single adjuvant agent in the management of early breast cancer: analysis at six years by Nolvadex adjuvant trial organisation. Lancet 1985; 1:836- 840

48. Ribeiro, G. and R. Swindell. The Christie Hospital tamoxifen (Nolvadex) adjuvant trial for operable breast carcinoma - 7 year results. Eur J Cancer Clin Oncol 1985; 21(8):897-900.

49. Taguchi, O. and Y. Nishizuka. Reproductive tract abnormalities in female mice treated neonatally with tamoxifen. Am J Obstet Gynecol 1985; 151:675-678.

50. Taylor, S.G. et al. Adjuvant CMFP versus CMFP plus tamoxifen versus observation alone in postmenopausal, node-positive breast cancer patients: three-year results of an eastern co- operative oncology group study. J Clin Oncol 1985; 3(2):144-154.

51. Product Monograph. TamofenR (tamoxifen citrate) tablets of 10 and 20 mg. Rhone-Poulenc Pharma Inc. Montreal, Quebec, January 13.1988.

52. Product Monograph. Nolvadex (tamoxifen citrate) Tablets 10 mg. Nolvadex-D (tamoxifen citrate) Tablets 20 mg. ICI Pharma, Mississauga, Ontario, June 14, 1988.

53. United States Pharmacopeia. Drug information for the health care provider. Eighth Edition 1988; IB:2005-2006.

54. Zelissen, P., M. A. Blankenstein and J. J. Tukker. Comparative bioavailability of tamoxifen citrate. Comparison of tamoxifen citrate GenericsR with Nolvadex-DR in 16 male volunteers. Data on file, Genpharm Inc., 1988.